This is a competing renewal of R01GM49202-09, which has for the past nine years supported the in-depth investigations of the molecular nature of general anesthetic interaction with the neuronal membrane constituents. Recent research efforts have combined the use of modern molecular biology techniques with various biophysical approaches, notably the state-of-the-art, high-resolution, solution- and solid-state NMR, to elucidate the effects of general anesthetics on the structures and dynamics of the transmembrane (TM) segments of the human glyeine receptors (GIyR). Unlike many conventional functional analysis that establishes the protein sequence-function relationship based on inferences from anesthetic sensitivity, this project aims at identifying the structure-function and dynamics-function relationships with direct binding and dynamics analysis at the sub-molecular and atomic levels. The support of this R01 grant has helped (1) to develop the NMR methods for non-destructive quantification of low-affinity anesthetic binding to receptor proteins in the membrane-mimetic environments; (2) to demonstrate that volatile general anesthetics as a molecule group distinguish themselves from the structurally similar nonimmobilizers (nonanesthetics) by their ability to preferentially target the amphipathic sites at the membrane interfacial regions; (3) to illustrate with many lines of experimental evidence that specific binding of volatile anesthetics to TM channels can lead to functional changes without necessarily causing secondary structural changes; (4) to conceptualize a testable hypothesis that the global changes in membrane protein dynamics might underlie a common mechanism for general anesthetic action on a diverse range of anesthetic-sensitive proteins; and (5) to reason with experimental support that the role of interfacial water should not be ignored in analyzing the action of general anesthetics on TM channels. These new insights, which were not fully comprehended at the time of the last competitive renewal 3.5 years ago, will advance the research to a new level. Substantive new structural and dynamical data on the alpha1 subunits of the human GIyR (GlyRa1) have been obtained by the investigators to warrant the continuation of the project with the following four new specific aims: 1. To differentiate the effects of anesthetics and nonanesthetics on the water accessibility to the backbone arnide protons of the interfacial and structural residues; 2. To quantify the anesthetic and nonanesthetic effects on the tertiary structure of the TM2+TM3 domains of the human GlyRa1 subunit, along with the extracellular linker, in DPC micelles and in oriented lipid bilayers; 3. To determine the backbone dynamics of the WT TM2+TM3 and two mutants, TM2(S267Y)+TM3 and TM2+TM3(A288W), in DPC micelles in the presence and absence of volatile anesthetics; and 4. To characterize the anesthetic effects on the quaternary structure of the homopentameric channel composed of TM1+TM2+TM3 domains of human GlyRalpha1 subunit. These new studies will eventually relate the structurally specific molecular events to the functional consequences of the action of general anesthetics.